Please use this identifier to cite or link to this item:
|Title:||Part I：探討Evo00825對人類荷爾蒙不依賴型前列腺癌細胞之作用機轉;Part II：探討Pim-1在微小管作用劑引發的細胞凋亡中所扮演的角色|
Part I: Study of anticancer mechanism of Evo00825 in human hormone-refractory prostate cancer cells;Part II: Study of the role of Pim-1 in microtubule-binding agents mediated apoptosis in human hormone-refractory prostate cancer cells
|Publication Year :||2010|
|Abstract:||Evodiamine為由植物吳茱萸(Evodiae Fructus)分離之主成分，有研究指出其可抑制多種癌細胞的生長活性。我們實驗室獲得一系列Evodiamine衍生物，並利用SRB assay測試其抗癌活性，發現當中有很多的衍生物都比Evodiamine強10倍，其中Evo00825在眾多的衍生物中最為突出。其可抑制多種癌細胞的生長活性，包括攝護腺癌細胞(PC-3、DU145與LNCaP)、肝癌細胞(Hep3B)、乳癌細胞(MCF-7)及大量表現P-glycoprotein的乳癌細胞(NCI/ADR-RES)，其IC50約介於50至210 nM之間。由於Evo00825也抑制了NCI/ADR-RES的細胞生長，故此化合物並非為P-glycoprotein的受質。隨著時間的增加，Evo00825會造成PC-3細胞產生G2/M期停滯，並於長時間下引起細胞凋亡。根據in vitro與in vivo微小管聚合試驗與免疫螢光試驗顯示，Evo00825造成PC-3細胞不正常有絲分裂紡錘體的產生。此化合物增加cyclin B1與MPM2的表現量，使Cdk1之Thr161位置與Cdc25C之Thr48位置磷酸化，同時也造成MAP4的磷酸化，這些結果顯示Evo00825使細胞停滯於有絲分裂期。此外，與粒線體相關的訊息傳遞也受到Evo00825之影響，包括Bcl-2與Bcl-xL之磷酸化、caspase-9與-3之活化及poly(ADP-ribose) polymerase (PARP)之斷裂。另外JNK抑制劑SP600125可顯著的抑制Bcl-2與Bcl-xL的磷酸化及caspase-3和PARP的斷裂，同時也阻止細胞進入有絲分裂期並降低Evo0025引起的細胞死亡。總結來說，這些結果顯示經由Evo00825引起的抗生長活性與細胞凋亡作用，是透過抑制微小管聚合、引發JNK相關的路徑，使細胞停滯在有絲分裂期及引起微小管調控之細胞凋亡途徑達到細胞死亡。
Pim-1為一種serine/threonine kinase的oncogene，負責參與細胞生長、增生與分化等多種功能，並對於攝護腺癌尤其是荷爾蒙不依賴型攝護腺癌的發生過程有密切的關係。第二篇主要探討Pim-1在微小管結合藥物於荷爾蒙不依賴型攝護腺癌細胞引發的細胞凋亡所扮演的角色，利用多種微小管結合藥物包括paclitaxel、vincristine、colchicine、evodiamine與Evo00825進行探討，結果顯示這些微小管結合藥物可透過活化轉錄因子STAT3引起Pim-1表現量的增加，利用Pim-1 siRNA可以使細胞對微小管結合藥物所產生的細胞毒性更為敏感並降低藥物所引起的G2/M期累積。根據免疫螢光試驗顯示Pim-1與微小管聚集在一起，但由微小管聚合試驗發現抑制Pim-1的表現並沒有改變微小管結合藥物所引起的微小管動態平衡。同時抑制Pim-1會減緩DNA damage sensor中ATM與DNA-PKcs的活化，但卻引起DNA雙股斷裂指標蛋白H2A.X之Ser139位置磷酸化的增加，有趣的是在comet assay中並沒有發現comet tail的增加，由此證實抑制Pim-1後細胞對於paclitaxel所增加的細胞凋亡與DNA損傷並沒有直接的關係。另一方面，根據免疫螢光試驗顯示抑制Pim-1會減少DNA-PKcs與Ku蛋白的結合，導致non-homologous end-joining (NHEJ)途徑受到抑制。總結來說，這些結果顯示Pim-1 knockdown可以抑制DNA-PKcs所參與的NHEJ途徑，使染色質保護受阻造成H2A.X的磷酸化，同樣也顯示Pim-1可以作為提高抗癌藥物活性的潛力標靶。
Evodiamine, a bioactive component derived from Evodiae Fructus, displays anticancer activity against a variety of types of human cancers. According to the anticancer potential of evodiamine, we obtained a series of evodiamine derivatives and determined their anticancer activities. After a screening test on cell proliferation by sulforhodamine B assay, we found several active derivatives demonstrating more than ten-fold potency compared with the parent compound. Accordingly, we studied the anticancer mechanism and evaluate the potential of Evo00825, which stood out from these derivatives. Evo00825 inhibited cell proliferation of various cancer cell lines, including prostate cancer PC-3, DU-145 and LNCaP, hepatocellular carcinoma Hep3B, breast cancer MCF-7 and P-glycoprotein (P-gp)-overexpressed NCI/ADR-RES cells with IC50 values ranging from 84 to 210 nM. The data also indicated that Evo00825 was a poor substrate for P-gp, since over-expressed P-gp did not blunt Evo00825-mediated effect. The cytofluorometric analysis showed that Evo00825 induced G2/M arrest of the cell cycle in a time-dependent manner, followed by a sequential increase of hypodiploid phase (apoptosis). Evo00825 inhibited microtubule assembly in both in vitro turbidity assay and in vivo microtubule spin-down experiment. Immunochemical examination showed that Evo00825 caused abnormal mitotic spindles in PC-3 cells. This compound also activated the up-regulation of cyclin B1 expression, Cdk1 phosphorylation at Thr161, Cdc25C phosphoryltaion at Thr48 as well as MAP4 phosphorylation and elevation of MPM-2 levels. These data indicated the occurrence of mitotic arrest to Evo00825 action. Moreover, several mitochondria-mediated intrinsic pathways were activated by Evo00825, including phosphorylation of Bcl-2 and Bcl-xL, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). The JNK inhibitor, SP600125, significantly blocked Evo00825-induced Bcl-2 and Bcl-xL phosphorylation, caspase-3 activity and PARP cleavage. It also abolished the mitotic arrest and rescued the apoptotic cell death caused by Evo00825. In summary, the data suggest that Evo00825 induces anti-proliferative and apoptotic effects in cancer cells through the inhibition of tubulin polymerization and stimulation of JNK activity, resulting in the induction of mitotic arrest and mitochondria-mediated apoptotic pathways and, ultimately, leading to apoptosis of cancer cells. The data also provide evidence supporting the further development of evodiamine derivatives.
Pim-1, a serine/threonine kinase functions as an oncogene, has diverse biological roles in cell survival, proliferation and differentiation and has been implicated in prostate cancer progression, particularly in hormone-refractory prostate cancers. In this study, we tried to identify the role of Pim-1 in microtubule-binding agent-mediated apoptosis in human hormone-refractory prostate cancer cells. Several microtubule-binding agents were used including paclitaxel, vincristine, colchicine and evodiamine. These agents activated STAT3 phosphorylation and its transcriptional activity, leading to up-regulation of Pim-1 expression. The knockdown of Pim-1 expression by siRNA transfection technique sensitized cells to the cytotoxic effects and reduced accumulation of G2/M phase of the cell-cycle caused by these microtubule-binding agents. Confocal microscopic examination showed that Pim-1 was co-localized with tubulin. However, the tubulin polymerization assay revealed that the knockdown of Pim-1 did not change the tubulin/microtubule dynamics under the treatment of microtubule-binding agents. The knockdown of Pim-1 delayed and reduced the activation of ATM and DNA-PKcs, a DNA damage sensor, but showed the activation of the phosphorylation of H2A.X at Ser139, a DNA double-stranded break marker, in an ATM-independent manner. Notably, the comet assay did not show an increase of comet tail suggesting that the sensitization of paclitaxel-mediated apoptosis by Pim-1 knockdown was not through the direct effect on DNA damage. Moreover, confocal microscopic examination showed that Pim-1 knockdown reduced the binding of DNA-PKcs and Ku protein, resulting in the inhibition of NHEJ pathway. In summary, we suggest that Pim-1 knockdown potentiates paclitaxel-induced H2A.X phosphorylation through the inhibition of Pim-1-involved and DNA-PKcs-signaled NHEJ pathway, leading to the blockade of rescuing chromatin defects. The data also reveal that Pim-1 kinase serves as a potential target to enhance the anticancer activity of therapeutic drugs.
|Appears in Collections:||藥學系|
Files in This Item:
|2.71 MB||Adobe PDF|
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.